Nucleosidylphosphite-borane compounds and method of making the same

ABSTRACT

Nucleosidyl phosphite-borane compounds of the formula: ##STR1## wherein: Nucleoside is a natural or synthetic nucleoside connected to the phosphorus atom via a hydroxyl oxygen; each X is independently selected from O and BHR 3  R 4  ;R 1  is selected from H, alkyl, aryl, alkyaryl, monovalent metal ions, and ammonium cation; R 2  is selected from OR 1  and N(R 5 ) 2 , wherein R 5  is independently selected from H, C 1  -C 10  linear or branched alkyl or aryl; R 3  is selected from H, CN, COOH, carboxyl salts, COOR 6  and CONHR 6 , wherein R 6  is selected from H, C 1  -C 10  alkyl, alkylaryl and aryl; R 4  is selected from H and C 1  -C 10  alky; and n is 1 to 2.

GOVERNMENT RIGHTS IN INVENTION

The invention may be used by the U.S. Government for Governmentalpurposes without the payment of royalties to the inventors.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of U.S. application Ser. No. 07/701,682filed May 10, 1991 now U.S. Pat. No. 5,143,907.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to phosphite-borane derivatives withnucleoside substituents that exhibit antineoplastic,anti-hyperlipidemic, and anti-inflammatory activity.

2. Description of the Related Art

A. Lewis base-borane Compounds

Various boron-containing compounds have previously been shown to exhibittherapeutic biological activity. For example, amine-borane compoundssuch as amine.BH₂ COOH, amine.BH₂ COOMe and amine.BH₂ CONHR have beendemonstrated to exhibit antitumor, anti-inflammatory and hypolipidemicactivities. Additionally, phosphite-borane compounds have been used inhydroboration under mild conditions (Pelter, A., et al, J. Chem. Soc.Chem. Commun. 1981, 1014). Since the first reports of phosphite-boranecompounds and their properties (Reetz, T., J. Am. Chem. Soc. 1960, 82,5039), very few phosphite-borane compounds have been synthesized and/orhad their properties investigated (Das, M. K., et a.1 Synth. React.Inorg. Met. Org. Chem. 1986, 16, 67; Martin, D. R. et al; Pennington, B.T., J. Inorg. Nucl. Chem. 1978, 40, 9; and Mutterties, E. L., "TheChemistry of Boron and its Compounds," Wiley, New York, 1967).

Generally, phosphite-borane derivatives may be considered as analogs ofalkylphosphates, (RO)₃ P═O vs. (RO)₃ PBH₃, as well as analogs ofalkylphosphonates, e.g., (RO)₂ P(O)CH₃ vs. (RO)₂ p(O)BH₃, or (RO)₂p(O)CH₂ X vs. (RO)₂ P(O)BH₂ X, wherein R is alkyl and X is heteroatomsubstituent. Since phosphate and phosphonate groups are present in avariety of biologically important molecules, e.g., DNA, RNA,phospholipids, aminophosphonates, etc., their boron-containing analogsmay prove useful as biomolecular probes and as potential therapeuticagents.

Additionally, several synthetic phosphonates, e.g., phosphonoaceticacid, phosphonoformic acid, etc., have been found to possess significantantiviral activity (Mayer, R. F., et al, Antimicrob. Agents Chemother.1976, 9, 308; Oberg, B., Pharmac. Ther., 1983, 19, 387; and Clerq, E.D., J. Med. Chem. 1986, 29, 1561). This antiviral activity coupled withthe established pharmacological activity of amine-borane derivativesmakes phosphite-borane derivatives potentially significant as a class ofbioactive compounds.

B. Modified Nucleotides

Ribo- and deoxyribonucleoside 5'-mono-, di-, and triphosphates play acentral role in the metabolism of nucleic acids, one of the mostimportant polymer molecules of living systems. It has long been realizedthat chemically modified analogs of nucleoside mono-, di-, andtri-phosphates may be useful tools to probe different steps of nucleicacid metabolism. It has also been recognized that they may have valuablechemo-therapeutic properties. Therefore, synthesis and study ofnucleotide analogs has long been in the center of interest.

Several modifications of the phosphate group have been carried out andthe derivatives are shown in Table 1 below. These derivatives mainlyinvolve phosphorothioates (Eckstein, F. Angew, Chem. Int. Ed. Engl.1983, 22, 423-439 and references therein, Eckstein, F. Ann. Rev.Biochem. 1985, 54, 367-402 and references therein, Ludwig, J.; Eckstein,F. J. Org. Chem. 1989, 54, 631-635, and Ludwig, J.; Eckstein, F. J. Org.Chem. 1991, 56, 5860:-5865), phosphorodithioates (Ludwig, J.; Eckstein,F. J. Org. Chem. 1991, 56, 1777-1783), phosphoramidates (Chambers, R.W.; Moffatt, J. G., J. Am. Cham. Soc. 1958, 80, 3752-3756; Chambers, R.W. et al, ibid, 1960, 82, 970-975; Moffatt, J. G.; Khorana, H. G., ibid,1961, 83, 649-658; Cramer, F. et al, Chem. Ber. 1961, 94, 1612-1621;Schaller et al, ibid, 1961, 94, 1621-1633; Cramer, F.; Neunhoffer, H.,ibid, 1962, 95, 1664-1669; Simoncsits, A.; Tomasz, J., Tetrahedron Lett.1976, 3995-3998; Tomasz, J.; Simoncsits, A., J.Carbohydrates-Nucleosides-Nucleotides 1978, 5, 503-522; Tomasz, J.,Nucleosides & Nucleotides 1983, 9, 63-79; Bakina, G. T. et al Bioorg.Khim, 1975, 1, 611-615 and Zarytora, V. F. et al, ibid 1975, 1,793-798), phosphonates (Anand, N.; Todd, A. R., J. Chem. Soc. 1951,1867-1872; Engel, R. Chem. Revs. 1977, 77, 349,-367 and referencestherein and Myers, T. C.; Simon, N. L., J. Org. Chem. 1985, 30,443-446), phosphorofluoridates (Wittmann, R., Chem. Ber. 1963, 96,771-779, Johnson, P. W. et al Nucleic Acids Res. 1975, 2, 1745-1749;Staley, B.; Yount, R. G., Biochemistry 1972, 11, 2863-2871 and Eckstein,F. et al, ibid, 1975 114, 5225-5232), phosphates or H-phosphonates(Corby, N. S. et al J. Chem. Soc., 1952, 3669-3674, Sir Todd, A., ibid,1961, 2316-2320 and Holy, A. et al Cell. Czech. Chem. Commun. 1965, 30,1635-1641), phosphorazidates (Chladek, S. et al, Biochemistry 1977, 16,4312-4319), phosphonoselenoates (Sekine, M.; Hata, T., Tetrahedron Lett.1979, 801-802) and alkyl phosphates (Hoffmann, P. J.; Blakley, R. L.,Biochemistry 1975, 14, 4804-4812.

                                      TABLE 1                                     __________________________________________________________________________    Structural formulae of nucleoside 5'-mono-, di- and                           triphosphates derivatised at the phosphorus                                    ##STR2##                                                                                  ##STR3##                                                                                 ##STR4##                                              X              Y   X      Z    Y   X                                          __________________________________________________________________________     1                                                                              S.sup.-    2 O.sup.-                                                                           S.sup.-                                                                            4 O.sup.-                                                                            O.sup.-                                                                           S.sup.-                                    10                                                                              NH.sub.2, NHR' or NR'.sub.2                                                              3 S.sup.-                                                                           O.sup.-                                                                            5 O.sup.-                                                                            S.sup.-                                                                           O.sup.-                                    13                                                                              CH.sub.3 or CH.sub.2 R'                                                                 14 CH.sub.3                                                                          O.sup.-                                                                            6 S.sup.-                                                                            O.sup.-                                                                           O.sup.-                                    15                                                                              F         16 F   O.sup.-                                                                            7 O.sup.-                                                                            S.sup.-.sub.3                                                                     S.sup.-                                    18                                                                              H         20 N.sub.3                                                                           O.sup.-                                                                            8 S.sup.-                                                                            O.sup.-                                                                           S.sup.-                                    19                                                                              N.sub.3              11 O.sup.-                                                                            O.sup.-                                                                           NH.sub.2                                   22                                                                              Se.sup.-             12 R'NH O.sup.-                                                                           O.sup.-                                     ##STR5##              17 21 23                                                                         F N.sub.3 R'O                                                                      O.sup.- O.sup.- O.sup.-                                                           O.sup.- O.sup.- O.sup.-                    __________________________________________________________________________

Derivatization at the phosphorus moiety by replacing a non-bridgingoxygen atom confers chirality on the P1 phosphorus of compounds 2, 4, 7,8, and 11 as well as P2 of 5 and 7. As a result of the chirality of thesugar residue, nucleotide 5'-di- and triphosphate derivatives 2, 4, 5,7, 8 and 11 formed during chemical synthesis exist as pairs ofphosphorus, Rp and Sp, diastereoisomers. The chirality of phosphorusrenders these derivatives suitable tools for studying thestereochemistry of enzyme catalyzed reactions. The diastereoisomers,however, have to be separated, since the diastereoisomeric purity of thesubstrate is an essential prerequisite for stereochemical studies.

Taking into consideration the chirality of phosphorus, it is notsurprising that, among the nucleoside 5'-mono-, di-, and triphosphateanalogs listed in Table 1 above, the thiophosphates have foundwidespread applications in biochemistry and molecular biology.Nucleoside phosphorothioate diastereoisomers have been used to determinethe stereochemical course of numerous enzyme catalyzed nucleotidyl andphosphoryl transfer reactions (Eckstein, F., Angew Chem. Int. Ed. Engl.1983, 22, 423-439 and references therein and Eckstein, F. Ann. Rev.Biochem. 1985, 54, 367-402 and references therein). The stereochemicaloutcome of an enzymic reaction, i.e., whether it proceeds with inversionor retention of configuration at phosphorus, is an informative criterionabout the presence or absence of a covalent enzyme intermediate. The Spdiastereoisomers of 4, as substrates of RNA and DNA polymerases, havesuccessfully been employed for sequencing (Gish, G.; Eckstein, F.,Science, 1988, 240, 1520-1522 and Nakamaye, K. L. et al, Nucleic AcidsRes. 1988, 16, 9947-9959), oligonucleotide-directed mutagenesis(Nakamaye, K. L.; Eckstein, F., Nucleic Acids Res., 1986, 14, 9679-9698and Sayers, J. R. et al, ibid, 1988, 16, 791-802), and the labeling ofthe hybridization probes (Haase, A. T. et al, Science 1985, 227, 189-192and Bahmanyar, S. et al, Science, 1987, 237, 77-80). Triphosphatederivatives 6 are also substrates for polymerases (Smith, M. M. et al,Biochemistry 1978, 17, 493-500). Many enzymes show strong preference foreither the Sp or Rp diastereoisomer of triphosphates 4 and 5. Forexample, of guanine nucleotide-binding proteins (G-proteins) which areimplicated in signal transduction pathways (Bourne, H. R. et al, Nature,1990, 348, 125-132), transducin (the G-protein involved in vision) has astronger affinity for the (Sp)-guanosine 5'-O-(2-thiotriphosphate). Onthe other hand, the G-protein responsible for the oscillatory release ofCa²⁺ ions in most cells is preferentially activated by the(Rp)-diastereoisomer (von zur Muhlen, R.; Eckstein, F.; Penner, R. Proc.Acad. Sci. U.S.A. 1991, 88, 926-930). The stereoselectivity of theenzymes can be reversed by changing the metal cation necessary for theenzyme action from a hard to a soft one (Armstrong, V.; Eckstein, F.,Eur. J. Biochem., 1976, 70, 33-38).

Nucleoside-boranophosphates and boranophosphoramidates (phosphite-boranecompounds), the compounds of the present invention, may be considered asanalogs of corresponding phosphates or thiophosphates, where the oxygenor sulfur has been replaced with a borane substituent. These derivativeshave similar charges and thus resemble phosphates or phosphorothioates.

On the other hand, differences are expected between boranophosphates andthiophosphates in reactivity, hydrogen bonding and metal ion chelatingability which may be a determinant for enzyme reactions. Consequently,it seems reasonable to suppose that boranophosphates may find similarand, at the same time, complementary applications to thiophosphates 1-6.Our initial results support this supposition.

The thymidine 5'-O-(1-boranotriphosphate) can substitute for thymidine5'-triphosphate (dTTP) in the extension of a deoxyribo 17-mer primer bySequenase, a modified T7 DNA polymerase, using a 25-mer templatecontaining one 2'-deoxyadenosine residue. No detectable pause inpolymerization was found at the dTTP incorporation site. These findingssuggest that thymidine 5'-0-(1-boranotriphosphate), possibly one of thetwo phosphorus diastereoisomers like the (Sp) diastereoisomer of theanalogous thiotriphosphates 4 (Burgers, P. M. J.; Eckstein, F., J. Biol.Chem. 1979, 254, 6889-6893 and Romaniuk, P. J.; Eckstein, F., ibid 1982,257, 7684-7688) is a substrate for polymerases.

It was also observed that acid phosphatase from sweet potato (EC3.1.3.2) and 5'-nucleotidase from Crotalus adamanteus venom (EC 3.1.3.5)completely hydrolyses thymidine 5'-boranophosphate to thymidine. On theother hand, thymidine 5'-boranophosphate is a very poor substrate (or aninhibitor) of alkaline phosphatase from Eschrichia coli (EC 3.1.3.1).The analogous thiophosphate, thymidine 5'-thiophosphate, is acompetitive inhibitor of both acid and alkaline phosphatases. The factthat thymidine 5'-boranophosphate is a substrate of acid phosphatase,while thymidine 5'-thiophosphate is a competitive inhibitor of the sameenzyme, is remarkable and demonstrates the potential for thecomplimentary use of boranophosphates and thiophosphates to study thedetails of the mechanism of enzymic reactions.

In addition to molecular biology studies, modified nucleosides andnucleotides have demonstrated considerable pharmacological activity inthe antiviral and antitumor areas (Mitsuya, H. Broder, S., Proc. Natl.Acad. Sci. USA, 1986, 83, 1911.-1915; Mitsuya, H. et al, Proc. Natl.Acad. Sci. USA 1985, 82, 7096-7100; Lin, T. S. et al, J. Med. Chem.,1988, 31, 336-340; Beauchamp, L. M. et al, J. Med. Chem. 1988, 31,144-149; Remy, R. J., Secrist III, J. A., Nucleosides Nucleotides 1985,4, 411-427; Prusoff, W. H., Ward, D. C., Biochem. Pharmacol., 1976, 25,1233-1239; Marquez, V. E. et al, J. Med. Chem. 1988, 31, 1687-1694; Lin,T. -S., Prusoff, W. H., J. Med. Chem. 1978, 21, 109-112; Johnson, F. etal, J. Med. Chem. 1984, 27, 954-958; Secrist et al, J. Med. Chem. 1988,31, 405-410; Farquherz, D., Smith, R., J. Med. Chem. 1985, 28,1358-1361; Hunston, R. H. et al, J. Med. Chem. 1984, 27, 440- 444;Farquher, D. et al, J. Med. Chem. 1983, 26, 1153-1158; McGuigan et al,Nucleic Acids Res. 1989, 17, 6065-6075; McGuigan et al, Nucleic AcidsRes. 1989, 17, 10171-10177; Colin, B. et al Nucleic Acids Res. 1989, 17,7195-7201 and Lambert et al, J. Med. Chem. 1989, 32, 367-374). Thus,phosphite-boranes with nucleoside substituents may combine thepharmacological properties of Lewis-base-borane compounds and those ofmodified nucleosides to give superior therapeutic agents.

While it is clear that considerable potential exists for the utility ofphosphate-borane derivatives with nucleoside substituents asbiomolecular probes and therapeutic agents, it is equally dear that notmuch effort has been focused on exploiting this potential. The presentinvention arose from our ongoing research into boron analogs ofbiomolecules potentially useful as probes and therapeutic agents.

It therefore is an object of the present invention to provide newphosphite-borane derivatives including active antineoplastic,antihyperlipidemic, and anti-inflammatory agents.

It is another object of the present invention to provide new processesfor synthesizing phosphate-borane derivatives exhibiting antineoplastic,anti-hyperlipidemic, and anti-inflammatory activity.

Other objects and advantages will be more fully apparent from theensuing disclosure and appended claims.

SUMMARY OF THE INVENTION

The phosphate-borane compounds of the present invention comprise acategory of phosphite-borane compounds within the broad scope of thephosphate-borane compounds of our prior copending U.S. application Ser.No. 07/701,682 filed May 10, 1991, wherein the phosphate moietysubstituents comprise nucleosidyl substituent. An additional categoryrepresents simple phosphite-BH₃ compounds where phosphate is a mono- ordialkyl-phosphite or its salts. These compounds also possess significantantitumor, anti-hyperlipidemic and anti-inflammatory activities. Inaddition, these compounds are useful for studying enzymatic processes atthe molecular level.

The phosphate-borane compounds of the present invention correspond tocompounds of the following general categories:

a. A phosphate-borane compound corresponding to the formula ##STR6##where: R₁ is independently selected from H, C_(1-C) ₂₀ alkyl, alkylaryl,aryl, and trialkylsilyl, with the proviso that both R₁ groups cannotsimultaneously be H₁ ; and

R₂ is selected from H, and a monovalent cation such as Li⁺, Na⁺, K⁺, NH₄⁺, and N(R₃ ³)₄, where R₃ is independently selected from H, C₁ -C₂₀alkyl.

b. A phosphate-borane compound corresponding to the general formula##STR7## where: nucleoside is a natural or synthetic (e.g. ribo-,deoxyribo-, dideoxyribo-, arabino-, xylo, acyclic, carbocyclic,oxetanocin, etc.) nucleoside connected to the phosphorus via one of itshydroxyl oxygens;

R₁ is selected from H, alkyl, aryl, alkylaryl, monovalent metal ions,and an ammonium cation;

R₂ is selected from OR₁, where R₁ is as above, PO₄ ²⁻, P₂ O₇ ³⁻,OP(O)(OR₅)₂, OPO₃ (OR₅)₃, and N(R₅)₂, where R₅ is independently selectedfrom H, C₁ -C₁₀ linear or branched alkyl, and aryl;

R₃ is selected from H, CN, COOH, carboxyl salts, COOR₆ and CONHR₆,wherein R₆ is selected from H, C₁ -C₁₀ alkyl, alkylaryl and aryl; and

R₄ is selected from H and C₁ -C₁₀ alkyl.

c. A phosphate-borane compound corresponding to the formula ##STR8##where: nucleoside, R₁, R₃ and R₄ are the same as in category b;

X=O or BHR₃ R₄ ;

R₂ is selected from OR₁, PO₄ ²⁻, OP(O)(OR₅)₂ and N(R₅)₂, wherein R₅ isindependently selected from H, C₁ -C₁₀ linear or branched alkyl, andaryl.

d. A phosphate-borane compound corresponding to the formula ##STR9##where: Nucleoside, R₁, R₃ and R₄ are the same as in category b;

X is independently selected from O and BHR₃ R₄ ;

R₂ is selected from OR₁ and N(R₅)₂, wherein R₅ is independently selectedfrom H, C₁ -C₁₀ linear or branched alkyl, and aryl.

Another aspect of the present invention relates to processes forpreparing phosphate-borane compounds of the above formulae. It has beendiscovered that phosphate-borane compounds of these types can beprepared i) from a nucleoside by boranophosphorylation in a multistepprocess, ii) from nucleoside substituted phosphates by boronation in amultistep process or iii) by condensation of a di- ormono-alkylphosphite-borane with a nucleoside. The final productsgenerated by these processes, as well as the intermediate boronatedproducts of the multistep processes, comprise phosphate-borane compoundsof the present invention. The phosphate-borane compounds of class (a.)above may be prepared by hydrolysis of trialkylphosphite-boranes.

Yet another aspect of the present invention relates to the use of thesecompounds to treat tumors, inflammation and hyperlipidemia.

DETAILED DESCRIPTION OF THE INVENTION

The novel phosphate-borane derivatives of the present invention are afurther development of the phosphate-borane compounds broadly disclosedin our prior copending application No. 07/701,682 filed May 10, 1991, inthat in the phosphate-borane compounds of the present invention, the inthat the phosphate is connected to a nucleoside or, in the case ofborane adducts of mono- or dialkyl-phosphites or their salts, the boranegroup represents BH₃.

The phosphate-borane compounds of the present invention correspond tothe following general categories:

a. phosphite-borane compounds corresponding to the formula: ##STR10##where: R₁ is independently selected from H, C₁ -C₂₀ alkyl, alkylaryl,aryl, and trialkylsilyl, with the proviso that both R₁ groups cannotsimultaneously be H₁, and

R₂ is selected from H, and a monovalent cation such as Li⁺, Na⁺, K⁺, NH₄⁺, N(R₃ ⁺)₄, where R₃ is independently selected from H, and C₁ -C₂₀alkyl.

b. phosphite-borane compounds corresponding to the general formula##STR11## where: Nucleoside is a natural or synthetic (e.g. ribo-,deoxyribo-, dideoxyribo-, arabino-, xylo, acyclic, carbocyclic,oxetanocin, etc.) nucleoside connected to the phosphorus via a hydroxyloxygen;

R₁ is selected from H, alkyl, aryl, alkylaryl, monovalent metal ions,and an ammonium cation;

R₂ is selected from OR₁, where R₁ is as above, PO₄ ²⁻, P₂ O₇ ³⁻,OP(O)(OR₅)₂, OPO₃ (OR₅)₃, and N(R₅)₂, where R₅ is independently selectedfrom H, C₁ -C₁₀ linear or branched alkyl, and aryl;

R₃ is selected from H, CN, COOH, carboxyl salts, COOR₆ and CONHR₆,wherein R₆ is selected from H, C₁ -C₁₀ alkyl, alkylaryl and aryl; and

R₄ is selected from H and C₁ -C₁₀ alkyl.

c. phosphite-borane compounds corresponding to the formula ##STR12##where: Nucleoside, R₁, R₃ and R₄ are the same as in category b;

X=O or BHR₃ R₄ ;

R₂ is selected from OR₁, PO₄ ²⁻, OP(O)(OR₅)₂ and N(R₅)₂, wherein R₅ isindependently selected from H, C₁ -C₁₀ linear or branched alkyl, andaryl.

d. phosphite-borane compounds corresponding to the formula: ##STR13##where: Nucleoside, R₁, R₃ and R₄ are the same as in category b;

X is independently selected from O or BHR₃ R₄ ;

R₂ is selected from OR₁ and N(R₅)₂, wherein R₅ is independently selectedfrom H, C₁ -C₁₀ linear or branched alkyl, and aryl.

In the phosphite-borane compounds of the foregoing formulae, the alkylmoiety, whether itself or as a part of alkylaryl or aryl radicals orammonion ions, may either be linear or branched.

It is to be appreciated that the aliphatic and/or aromatic substituentsreferred to above may optionally be substituted with heteroatoms orotherwise further substituted, subject to the proviso that such furthersubstitution does not preclude the utility of the resulting compound.

The nucleoside preferably is selected from a ribo-, deoxribo-,dideoxyribo-, arabino- or an acyclic-nucleoside; R₁ is selected from H,C₁ -C₁₀ alkyl, monovalent-ions Li⁺, Na⁺, K⁺, NH₄ ⁺ and (n-C₄ H₉)₃ NH⁺ ;R₂ is OR₁, PO₄ ²⁻, P₂ O₇ ³⁻ or NR₅)₂ where R₅ is independently selectedfrom H or C₁ -C₅ linear or branched alkyl; R₃ is selected from H, CN,COOH, COOCH₃ and CONHC₂ H₅ ; and R₄ is H.

In a particularly preferred aspect, nucleoside is a ribo- or a deoxyribonucleoside; R₁ is H, CH₃, CH₂ CH₃ or CH₂ CH₂ CN; R₂ is OR₁, PO₄ ²⁻, P₂O₇ ³⁻ or N(i-C₃ H₇)₂ ; R₃ is H, or CN; and R₄ is H.

Exemplary phosphite-borane derivatives of the present invention includethe following:

Diethylphosphite-borane

Diethylphosphite-borane, sodium salt

Dibutylphosphite-borane, tetra-n-butylammonium salt

Ethyl(isopropyl)phosphite-borane, sodium salt

Dimethylphosphite-borane, ammonium salt

Mono-n-butylphosphite-borane, lithium salt

Mono-i-propylphosphite-borane

Monomethylphosphite-borane, sodium salt

Thymidine-5'-boranophosphate

2'-Deoxyadenosine-3'-boranophosphate

2'-Deoxyguanosine-5'-(N,N-diisopropyl)boranophosphoramidate

Thmidine-5'-O-((α-boranodiphosphate)

2'-Deoxycytidine-5'-O-(α-boranotriphosphate)

Cytidine-5'-O(α,β-diboranodiphosphate)

Guanosine-5'-O-(α,β-diboranotriphosphate)

Adenosine-5'-O-(α,γ-diboranotriphosphate)

Xanthosine-5'-O-(α,β,γ-triboranotriphosphate)

2'-Deoxyinosine-5'-O-(β-boranotriphosphate)

ara-Adenosine-5'-O-(α,γ-diboranotriphosphate)

Acyclovir-0-(β,γ-diboranotriphosphate)

Ribavirin-5'-O-(α,β-diboranodiphosphate)

Adenosine-2'-boranophosphate

Uridine-5'-O-(β-borano-β-diisopropylamino-β-[2-cyano]ethoxy-diphosphate)

2'-Deoxycytidine-5'-O-(β-borano-β-diisopropylamino-β-methoxy diphosphate

Guanosine-5'-boranophosphate

Uridine-2'-boranophosphate

Cordycepin-2'-boranophosphate

2'-Deoxycytidine-3'-boranophosphate

Adenosine-3'-boranophosphate

Cytidine-5'-(N,N-diisopropyl)boranophosphoramidate

Guanosine-5'-O-(α-boranodiphosphate)

Ribavirin-5'-O(α-boranodiphosphate)

Thymidine-5'-O-(β-boranodiphosphate)

2'-Deoxyguanosine-5'-O-(α,β-diboranodiphosphate)

2'-Deoxyadenosine-5'-O-(α,β-diboranotriphosphate)

ara-Adenosine-5'-O-(α-boranotriphosphate)

2'-Deoxyinosine-5'-O-(α-boranotriphosphate)

Inosine-5'-O-(α,β-diboranotriphosphate)

Uridine-5'-O-(α,β-diboranotriphosphate)

2'-Deoxyuridine-5'-O-(α,γ-diboranotriphosphate)

Guanosine-5'-O-(α,γ-diboranotriphosphate)

Adenosine-5'-O-(β-boranotriphosphate)

Cordycepin-5'-O-(β-boranotriphosphate)

2'-Deoxycytidine-5'-O-(γ-boranotriphosphate)

2'-Deoxyxanthosine-5'-O-(γ-boranotriphosphate)

Adenosine-5'-O-(β-boranodiphosphate)

Uridine-2'-boranophosphate

Cytidine-2'-boranophosphate

2'-Deoxyadenosine-5'-O-(β,γ-diboranotriphosphate)

Guanosine-5'-O-(β,γ-diboranotriphosphate)

Thymidine-5'-O-(α,β,γ-triboranotriphosphate)

2'-Deoxyuridine-5'-O-(α,β,γ-triboranotriphosphate)

The present invention also comprises a method for preparing thecompounds of the present invention. Four distinct processes have beenemployed to synthesize the compounds of the present invention.

The first multistep process which may be used to producephosphite-borane compounds of the present invention involves thefollowing basic steps:

a. phosphitylation of a nucleoside, a nucleoside phosphate, anucleosidediphosphate, a nucleoside boranophosphate, a nucleosideα-boranodiphosphate, a nucleoside β-boranodiphosphate or anucleoside-α,β-diboranodiphosphate.

b. transfer of boron from an appropriate Lewis base to thephosphoramidite intermediate,

c. de-esterification,

d. hydrolysis of boranophosphoramidate to boranophosphate or reactionwith orthophosphate or pyrophosphate to give the correspondingα-boranodiphosphate or α-boranotriphosphate respectively.

These steps are presented graphically in Scheme 1. ##STR14##

The second process which can be used for the preparation ofphosphite-borane compounds of the present invention also comprisesseveral steps and is shown in Scheme 2. ##STR15##

The steps involved are:

a. transfer of borane from an appropriate Lewis base-borane adduct tothe chloro(N,N-dialkyl)alkylphosphosramidite,

b. boranophosphorylation of the nucleoside, or an appropriate nucleosidephosphate,

c. de-esterification,

d. hydrolysis of borano-phosphoramidate to boranophosphate or reactionwith orthophosphate or pyrophosphate to give the correspondingα-boranodiphosphate or α-boranotriphosphate respectively.

The last two steps are the same as in the first process.

It should be noted that the above processes comprise individual reactionsteps which can be individually utilized to prepare a full range ofphosphate-borane derivatives of the present invention.

A third process which can be used for the preparation of thephosphite-borane derivatives of the invention is the condensation of amono- or dialkylphosphite-borane derivative with a nucleoside, e.g.,adenosine, guanosine, cytidine, thymidine, or uridine.

Finally, the borane (BH₃) derivatives of a monoalkyl- or adialkyl-phosphite may be prepared in one step by base hydrolyses of atrialkylphosphite.

The compounds of the present invention have pharmaceutical activity,including anti-inflammatory, anti-hyperlipidemic, and antineoplasticactivity, and are useful in treating mammals for inflammation,hyperlipidemia, and neoplasia conditions.

A method of combatting hyperlipidemia in an animal subject in need ofsuch treatment comprises administering to the animal subject ahyperlipidemia-combatting amount of a compound of the present invention.

A method of producing an anti-inflammatory response in an animal subjectin need of such treatment comprises administering to the animal subjectan inflammation-combatting amount of a compound of the presentinvention.

A method of combatting tumors, preferably solid tumors (e.g.,adenocarcinoma, bronchogenic carcinoma, osteosarcoma, epidermoidcarcinoma, breast carcinoma, glioma) in an animal subject in need ofsuch treatment comprises administering Lo the animal subject atumor-combatting amount of a compound of the present invention, afterwhich the tumor preferably is exposed to thermal (low energy neutrons)radiation in an amount effective for ¹⁰ B located in the tumor (byvirtue of the administration of the compound to the subject) to capturea neutron, decay, and release an alpha particle in cells of the tumor.

The above-described method of combatting tumors is a preferred modalityof anti-tumor treatment; however, in addition to such utility in boronneutron capture therapy, the compounds of the present invention alsohave inherent anti-tumor utility.

Specifically, the compounds of the present invention exhibit cytotoxicactivity against colorectal carcinoma, leukemia, osteosarcoma, gliomaand bronchogenic carcinoma, by functioning as antimetabolites.Correspondingly, the compounds of the present invention facilitate amethod of treating a tumor-bearing mammal, comprising of administeringto such mammal a therapeutically effective amount of a phosphite-boranecompound of the present invention.

Subjects to be treated by the methods of the present invention includeboth human and animal (e.g., bird, dog, cat, cow, horse) subjects, andare preferably mammalian subjects.

Animal subjects are administered compounds of the present invention at adaily dose of preferably at least about 0.1 mg/kg weight of the animalsubject, more preferably at least about 0.5 mg/kg, and most preferablyat least about 2 mg/kg. The daily dose is preferably not more than about1000 mg/kg, more preferably not more than about 200 mg/kg, and mostpreferably not more than about 50 mg/kg.

As noted above, the compounds of the present invention may beadministered per se or in the form of a pharmaceutically acceptablesalt. When used in medicine, the salts of the compounds of the presentinvention should be both pharmacologically and pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare the free active compounds or pharmaceuticallyacceptable salts thereof and are not excluded from the scope of thisinvention. Where appropriate, such pharmacologically andpharmaceutically acceptable salts include, but are not limited to ,those prepared from the following bases: sodium hydroxide, potassiumhydroxide, ammonium hydroxide, and calcium hydroxide.

The present invention also provides pharmaceutical formulations, bothfor veterinary and for human medical use, which comprise the activeagent (the compound of the present invention) together with one or morepharmaceutically acceptable carriers thereof and optionally any othertherapeutic ingredients. The carrier(s) must be pharmaceuticallyacceptable in the sense of being compatible with the other ingredientsof the formulation and not unduly deleterious to the recipient thereof.The active agent is provided in an amount effective to achieve thedesired pharmacological effect, as described above, and in a quantityappropriate to achieve the desired daily dose.

The formulations include those suitable for oral, rectal, topical,nasal, ophthalmic, or parenteral (including subcutaneous, intramuscularand intravenous) administration. Formulations suitable for parenteraladministration are preferred.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compounds into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into desired formulations.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets,or lozenges, each containing a predetermined amount of the activeingredient as a powder or granules; or a suspension in an aqueous liquoror a non-aqueous liquid, such as a syrup, an elixir, an emulsion, or adraught.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which optionally is mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent, or discharging agent. Molded tablets comprised of a mixture ofthe powdered active compound with a suitable carrier may be made bymolding in a suitable machine.

A syrup may be made by adding the active compound to a concentratedaqueous solution of a sugar, for example sucrose, to which may be addedany accessory ingredient(s). Such accessory ingredient(s) may includeflavorings, suitable preservatives, agents to retard crystallization ofthe sugar, and agents to increase the solubility of any otheringredient, such as a polyhydroxy alcohol, for example glycerol orsorbitol.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active compound, whichpreferably is isotonic with the blood of the recipient (e.g.,physiological saline solution).

Nasal spray formulations comprise purified aqueous solutions of theactive compound with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes.

Formulations for rectal administration may be presented as a suppositorywith a suitable carrier such as cocao butter, hydrogenated fats, orhydrogenated fatty carboxylic acids.

Ophthalmic formulations are prepared by a similar method to the nasalspray, except that the pH and isotonic factors are preferably adjustedto match that of the eye.

Topical formulations comprise the active compound dissolved or suspendedin one or more media, such as mineral oil, petroleum, polyhydroxyalcohols, or other bases used for topical pharmaceutical formulations.

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more accessory ingredient(s)selected from diluents, buffers, flavoring agents, binders,disintegrants, surface active agents, thickeners, lubricants,preservatives (including antioxidants), and the like.

The phosphite-borane compounds of the present invention, as well asother phosphite-borane compounds, may exhibit utility in antiviralapplications involving administration of such compounds to animal (humanor veterinary) subjects.

The following Examples are provided to illustrate the present invention,and should not be construed as limiti g the eof. Compounds areidentified in the first instance by a name and a reference number, andmay thereafter be identified solely by reference number, for ease ofreference.

Example 1 Diethylphosphite-borane, sodium salt; (compound 1)

Triethylphosphite-borane (0.70g) was taken with 1N NaOH (20 ml) and wasstirred until the oil was completely dissolved in aqueous layer. Theaqueous layer was washed with CH₂ Cl₂ (2×2 ml) and the water was removedin vacuo at room temperature. The residue was taken in ethyl acetate(˜20ml), allowed to stand for a few minutes and filtered. The filtratewas dried over anhydrous Na₂ SO₄, filtered and the solvent was removedto give pure product yield 0.59 g, 87.2%. ¹ H NMR.sub.(D20) : δ=3.79ppm, m, CH₂ ; 1.12 ppm, t, CH₃ ; 0.19 ppm, dq, J_(P),H =22±1Hz; J_(B),H=89±1Hz, BH₃. ¹¹ Bnmr.sub.(D20) : δ=-39.9 ppm, dq, J_(B),H =89±Hz,J_(P),B =147Hz. ³¹ Pnmr(D20): δ=91.1 ppm, J_(B),P =146±2Hz

Example 2 Thymidine-5'-borano(N,N-diisopropyl)phosphoramidate; (compound2)

3'-Acetylthymidine (284 mg, 1.0 mmol), 4-dimethylaminopyridine (24.5mg,0.2mmol) and diisopropylethylamine (0.7ml, 4.0 mmol) were dissolved inanhydrous CH₃ CN (10 ml) under Argon. To this solution, 2-cyanoethylN,N-diisopropylchlorophosphoramidite (0.275 ml, 1.3 mmol) was added andthe mixture was stirred for 1h. To the homogenous solutiondiisopropylethylamine-borane (1.74 ml, 10.0 mmol) was added. Afterstirring 12h at room temperature under argon atmosphere, the solvent wasremoved. The residue at evaporation was treated with a 1.5:1 mixture ofCH₃ OH/conc. NH₄ OH (25 ml) at RT for 5h. After evaportation,chromatography of the residue on a QA-cellulose [HCO₃ -] column(1.4×50.0 cm) by using a linear gradient of aqueous NH₄ HCO₃, pH=9.5(2L, 0-0.15M) and lyophilization of the appropriate fractions afforded320 mg of a white powder ofthymidine-5'-borano(N,N-diisopropyl)phosphoramidate, 2. ¹ H-NMR: δ=-0.079-0.739(2br. m, 3H; BH₃), 0.973, 0.981, 0.997, 1.021, 1.105 and1.127(6s, 12H; C(CH₃)₂), 1.764 and 1.772(2s, 3H; CCH₃), 2.142-2.244(m,2H; 2'-H), 3.269-3.402 (m, 2H; 5'-H), 3.720-3.795 (m, 2H; N-CH),3.973-4.027 and 4.375-4.455 (2m, 1H; 3'-H), 6.154 and 6.230(2t, ³J(H,H)=7.1 Hz, 1H; 1'-H), 7.481 and 7.519(2d, ⁴ J(H,H)=1.0 Hz, 1H; 6-H).³¹ p[¹ H]-NMR: δ=92.2 and 93.2 (2q, ¹ J(P,B)=147 Hz). ¹¹ B-NMR: δ=-36.8.TLC:R_(dTMp) =1.42.

Example 3 Thymidine-5'-boranophosphate; (compound 3)

Compound 2 (0.16 mg) was dissolved in 0.1N aqueous trifluoroacetic acid(40 ml). After 30 min. standing at room termperature, the solution wasevaporated and the residue was chromatographed on QA-cellulose [HCO₃ -]column (1.4×50 cm) by using a linear gradient of aqueous NH₄ HCO3,pH--9.5 (2L, 0-0.2M). Lyophilization of the appropriate peak gave 93 mg(52%) of a white solid as the product in the form of monohydrate of themonoammonium salt. ¹ H-NMR: δ=0.149(dq, ¹ I(B,H)=84.0 Hz, ² I(P,H)=21.6Hz, 3H; BH₃), 1.787(s, 3H; CH₃), 2.147-2.291(m, 2H; 2'-H),3.715-3.811(m, 2H; 5'-H), 3.978(unresolved, 1H; 4'-H), 4.424(unresolved, 1H; 3'-H), 6.182(t, ³ (H,H)=6.9 Hz, 1H; 1'-H), 7,690(s, 1H;6-H). ³¹ p[¹ H]-NMR: δ=79.3(q, ¹ I(P,B)=168 Hz). ¹¹ B-NMR: δ=-37.9(m).UV(H₂ O): γmax[nm](ε )maxγmin[nm]=268(9100) 236[pHs 2.0 and 7.0],268(7500)244[pH 11.0]. MS(electrospray ionization, 3000 V): m/z 321[3+3H]+.TLC:R_(dTMP) =1.25. RP-HPLC(C₁₈, A:0.02 M KH₂ PO₄, B:CH₃ CN,0-25% B/5 min, 3.0 mlmin⁻¹, t_(R) =4.98 min).

Example 4 Thimidine-5'-(α-borano-triphosphate),- (compound 4)

Compound 1 (160mg) was reacted with 0.5M (Bu₃ NH)₂ H₂ P₂ O7 in DMF (4.0ml) with the exclusion of atmospheric moisture at 55° C. for 4h. Themixture was poured into 0.1M aqueous NH₄ HCO₃ (40 ml) at roomtemperature, then separated on a DEAE-cellulose [HCO₃ -] column[2.1×52.0cm] by using a linear gradient of aqueous NH₄ HCO₃ (3L,0.1-0.3M, 10° C.). Appropriate fractions were combined and lyophilizedto give 61.2 mg (30%) of the product as a white solid. ¹ H-NMR:δ=-0.249-0.804(2 br.m, 3H; BH3), 1.780 and 1.788(2s, 3H; CH3),2.194-2.216(m, 2H; 2'-H), 3.961-4.168(2m, 3H; 4'-H, 5'-H), 4.449-4.497and 4.536-4.584(2m, 1H; 3'-H), 6.190(t, ³ I(H,H)=6.7 Hz, 1H; 1'--H),7.571 and 7.557(2s, 1H; H-6). ³¹ p[¹ H]-NMR: δ=-21.4(t, ² i(p,p)=21.4Hz, 1P; p²), -5.50(unresolved 1P; p³),82-84(br. m, 1P; p¹) ¹¹ B-NMR:δ=-38.6(m, ¹ i B,P)=137 Hz, ¹ I(B,H)=92 Hz). MS: m/z 498 [4+4H+NH₄ ].TLC: R_(dTTP) =1.40. RP-HPLC(C₁₈, A:0.2 M triethylammonium acetate,pH=7.5, B:methanol, 0-10% B/20 min, then 10% B/10 min, 4.0 mlmin-1,t_(R) =20.91(60%) and 24.57(40%) min.

Example 5 Adenosine 5'-boranophosphate; (compound 5)

To a 1.0M solution of THF-borane in THF (5.0 ml)(N,N-diisopropylamino)(cyanoethyl)phosphoramidic chloride (300 μl), 1.4mmol) was added under argon. After 10 min. stirring at RT, the solutionwas evaporated. The oily residue at evaporation was dissolved inacetonitrile (5 ml), and the slightly opalescent solution was added to amixture of 2', 3'-di-O-acetyladenosine (351 mg, 1.0 mmol),4-dimethylaminopyridine (24.5 mg, 0.2 mmol) and diisopropylethylamine(0.7 ml, 4.0 mmol) in acetonitrile (5 ml). The reaction mixture wasstirred with the exclusion of atmospheric moisture at RT for 3h. Conc.NH₄ OH (10 ml) was poured into the pale yellow solution under stirring.The solution was set aside at RT overnight, then evaporated to dryness.The evaporational residue was purified on a QA-cellulose [HCO₃ -] column(1.4×50.0 cm) by using a linear gradiant of aqueous NH₄ HCO₃, pH=9.5(2L, 0-0.15M) to give 138.0 mg of crude adenosine5'borano-(N,N-diisopropyl)phosphoramidate. The crude product was treatedwith 0.1M aqueous trifluoroacetic acid (25 ml) at RT for 30 min. Thesolution was evaporated and the residue was chromatographed on aQA-cellulose[HCO₃ -] column (1.4×50.0 cm) by using a linear gradient ofaqueous NH₄ HCO₃, pH 9.5 (2L, 0-0.25M). Appropriate fractions werepooled, concentrated to a small volume then lyophilized to give 49 mg(13%) of a white solid of the monoammonium salt of adenosine of5'-boranophosphate. UV, γ_(max) [nm] 258 (pH 2.0),260 (pHs 7.0 and11.0), γ_(min) [nm] 232 (pH 2.0),228 (pHs 7.0 and 11.0). ³¹ P-NMR:δ(ppm) 78.79 (q, ¹ I_(PB) =170 Hz) ¹ B-NMR: δ(ppm)-37 8 sextet,overlapping quartets of a doublet, ¹ I_(BP) =168Hz, ¹ I_(BH) =85Hz).

Example 6 Synthesis of3'-O-acetylthymidine-5'-diethylphosphite-cyanoborane; (compound 6)

3'-Acetylthymidine (0.35 g, 1.24 mmol), diethylphosphite-cyanoborane(0.22 g, 1.24 mmol) and dicyclohexylcarbodiimide, DCC, (2.48 mmol) weretaken in anhydrous acetonitrile and the mixture was stirred at roomtemperature for 48 hours. To the mixture another 2.48 mmol of DCC wasadded and the mixture was stirred for another 24 hours. After filtrationto remove insoluble materials, the solvent was removed under reducedpressure. The residue was taken in dichloromethane (40 ml) and waswashed with water (5×30 ml). The organic layer was dried, filtered, andconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel using EtOAc: hexane (9:1). Yield was 0.225g, 46.5%. 'H nmr (CDCl₃): δ=1.4,t and 1.44,t, 2CH₃ 's (OEt); 1.00-1.80,v. br., BH₂ ; 1.98 ppm, s and 1.99 ppm, s, CH₃ (for two diastereomers);2.13 ppm, s, CH₃ (OAc); 2.30-2.41, m, 2'CH₂ ; 4.15 ppm, m, 4'H; 4.25ppm, m, CH₂ 's (OEt); 4.29-4.47 ppm, m, 5'CH₂ ; 5.26 ppm, br., 3'H; 6.43ppm, m, 1'H; 7.36 ppm, 2 singlets, H6 (for two diastereomers) and 9.20ppm, s, NH. ¹³ C nmr (CDCl₃): δ=12.35 ppm, s, CH₃ (C₅); 16.02 and 16.10ppm, 2 doublets, CH₃ 's (OEt); 20.80 ppm, s, CH₃ (OAc); 36.54 ppm, s,2'CH₂ ; 65.19 and 65.33 ppm, 2 doublets, CH₂ 's (OEt); 65.94 ppm, d,5'CH₂ ; 74.21 ppm, s, 3'CH; 82.41 ppm, d, 4'CH; 84.06 ppm, s, 1'CH;112.10 ppm, C5; 134.69 ppm, s. C6; 150.61 ppm, s, C2; 163.61 ppm, s, C4;170.74 ppm, s, CO(OAc). ³¹ P nmr (CDCl₃): δ=94.26 ppm,. br.q., ^(1I)_(BP) =156 Hz (based on inner two peaks). FAB MS: MH⁺ : 444.3. Analysis,calculated: % C, 46.07; % H, 6.14; and % N, 9.48. Found: % C, 46.21; %H, 5.94 and % N, 9.37.

Example 7 Incorporation of Thymidine-5'-(α-borano-triphosphate) intooligonucleotides using polymerases

In vitro incorporation of thymidine-5'-α-boranotriphosphate into DNA wasstudied using two different polymerases; Sequenase (a modified T7 DNApolymerase; from USB) and the Klenow fragment of DNA polymerase I (fromNew England Biolabs). Both polymerases appeared to readily accept theboranophosphate nucleotide. The experiment was carried out using a17-mer primer extended against a 25-mer template containing a single dAcoding site. Extension was performed at 37° C. for 15 minutes in thepresence of 100 μm dATP, dGTP, dCTP and either 100 μM dTTP or 100α-borano-dTTP. The extension products were separated by denaturing PAGE.Autoradiography showed that the primer was extended to completionwithout any detectable pause in the presence of either normal orboronated dTTP.

Example 8 Inhibition of Alkaline Phosphatase Activity

2.0 absorbance (A260) units of thymidine-5'-boranophosphate and 0.5units of the enzyme alkaline phosphatase from E. Coli were taken in 40ml of 0.1M Na₂ CO₃ -NaHCO₃, pH 10.4 and the reaction was followed bytlc. No hydrolysis was observed even after 4h. Thethymidine-5'-phosphate, which does not have boron, is completelyhydrolyzed in <1h.

Example 9 Cytotoxic Activity of Phosphite-borane Compounds

The compounds prepared in accordance with the preceding Examples weretested for cytotoxic activity, by preparing a 1 mM solution of theadduct in 0.05% Tween® 80/H₂ O solution by homogenization. The resultingdrug solutions were sterilized by passage through an Acrodisc 45 μMsterilizer.

The following cell lines were maintained in accordance with literaturetechniques (literature source indicated parenthetically afteridentification of the cell line): murine L₁₂₁₀ lymphoid leukemia (Geran,R. I., et al, Cancer Chemotherapy Reports 1972, 3, 7-9); human Tmolt₃acute lymphoblastic T cell leukemia (Minowada, J., et al, J. Nat. CancerInt. 1972, 49, 891-895); colorectal adenocarcinoma SW480 (Liebovitz, A.,et al, Cancer Res. 1976, 36, 4562-4569); lung bronchogenic MB-9812(Aaronson, S. A., et al, Expt. Cell Res. 1970, 61, 1-5); osteosarcomaTE418 (Smith, H. S., et al, Int. J. Cancer 1976, 17, 219-234); KBepidermoid nasal pharynx (Geran, R. I., et al, Ibid.; Eagle, H., Proc.Soc. Expt. Biol. 1955, 89, 362-364); Hala-S³ suspended cervicalcarcinoma (Puck, T. T., et al, J. Exp. Med. 1956,103, 273-283); gliomaEH 118 Mg (Nelson-Rees, W. A., et al, Int. J. Cancer 1975, 16, 74-82)and Ileum HCT Colon.

The protocol used to assess cytotoxicity was that of Geran, et al,Cancer Chemotherapy Reports, 1972, 3, 7-9. Standards were determined ineach cell line. Values are expressed for the cytotoxicity of the drug asED₅₀ in μg/ml, i.e., the concentration which inhibits 50% of the cellgrowth determined by the trypan blue exclusion technique. Solid tumorcytotoxicity was determined by the method of Huang, E. S., et al, J.Pharm. Sci. 1972, 61, 108-110. Ehrlich ascites carcinoma in vivo tumorscreens were conducted in CF₁ male mice ≈28g) with test drugs at 8mg/kg/day I.P. by the method of Geran, et al (supra). 6-Mercaptopurinewas used as an internal standard.

The results of the cytotoxicity tests are set out in Table 1 below forcompounds 1, 3, 4, and 6, as well as 5FU, araC, hydroxyurea,cydoleucine, and 6MP.

                                      TABLE 1                                     __________________________________________________________________________    The Cytotoxic and Antitumor Activity of Phosphite-boranes                                    ED.sub.50 (μg/ml)                                                  % Inhibition                               Ileum                              in vivo        Colon       Osteo-                                                                             Lung       HCT                         Compound                                                                             Ehrlich Ascites                                                                       L.sub.1210                                                                       Tmolt.sub.3                                                                       SW480                                                                             KB HeLa-S.sup.3                                                                       Sarcoma                                                                            bronchogenic                                                                       Glioma                                                                              Colon                       __________________________________________________________________________    3              2.35                                                                             5.21            6.28            7.64                        4              2.52                                                                             3.49                                                        6      48      3.63                                                                             2.83                                                                              2.57                                                                              3.62                                                                             2.67 5.51 4.16 5.66                              5FU            1.41                                                                             2.14                                                                              3.09                                                                              1.25                                                                             2.47 --   5.64 1.28                              Ara C          2.76                                                                             2.67                                                                              3.42                                                                              2.54                                                                             2.13 --   7.24 1.88                              Hydroxyurea    2.67                                                                             3.18                                                                              4.74                                                                              5.29                                                                             1.96 7.57 7.33 2.27                              Cycloleucine   3.08                                                                             2.38                                                                              3.81                                                                              5.74                                                                             2.38 6.18 4.36 5.89                              6MP    99                                                                     __________________________________________________________________________

Example 10 Hypolipidemic Activity of Phosphite-borane Compounds

Test compounds (3 and 6) were suspended in an aqueous 1%carboxymethylcellulose solution, homogenized, and administered to CF1male mice (≈25 g) intraperitoneally for 16 days. On days 9 and 16, bloodwas obtained by tail vein bleeding, and the serum was separated bycentrifugation for 3 minutes. The serum cholesterol levels weredetermined by a modification of the Liebermann-Burchard reaction (Ness,A. T., et al, Clin. Chim. Acta. 1964, 10, 229-237). Serum was alsoanalyzed for triglyceride content by a commercial kit (BioDynamics/bmc)using a BMC single vial triglycerides colorimetric method 348201. Foodand water were available ad libitum for animals in the experiments.

In vitro enzymatic studies were determined using 10% homogenates of CF₁male mouse liver with compound 6. The enzyme activities were determinedby the following literature procedures (Chapman, J. M., Jr., et al, J.Med. Cham. 1979 22, 1399-1402); acetyl coenzyme A synthetase (Hoffmann,G., et al, Anal. Biochem. 1978, 84, 441-448); 3-hydroxy-3-methylglutarylcoenzyme A reductase (Haven, G. T., et al, J. Biochem. 1969, 65,171-175); acetyl coenzyme A carboxylase activity (Greenspan, M. D., etal, J. Biol. Chem. 1968, 243, 6373-6280); sn-glycerol-3-phosphate acyltransferase activity (Lamb, R. G., et al, Biochim. Biophys. Acta. 1977,489, 318-329); phosphatidylate phosphohydrolase activity (Mavis, R. D.,et al, J. Lipid Res. 1978, 19, 467-477); acyl CoA cholesterol acyltransferase (Balasubramaniam, S., et al, Eur. J. Biochem.1978, 90,377-383); and Squalene cyclase.

The results of the foregoing analytical tests are set out below in Table2 ("The Hypolipidemic Activity of Phosphite-boranes in CF₁ Mice at 8mg/kg/day I.P."), and Table 3 ("The Effects of Phosphite-boranes onEnzyme Activities of Lipid Metabolism of CF₁ Mice").

                  TABLE 2                                                         ______________________________________                                        The hypolipidemic activity of phosphite-boranes in CF.sub.1 mice at           8 mg/kg/day ip.                                                                      Percent of Control                                                            Serum Cholesterol                                                                          Serum Triglycerides                                       [N = 6] Day 9      Day 16   Day 16                                            ______________________________________                                        Control 100 ± 6 100 ± 5                                                                             100 ± 7                                        Compound                                                                      3       54         41       60                                                6       77         48       73                                                ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Effect of phosphite-boranes on mouse hepatic enzyme activities                IC.sub.50 × 10.sup.5 M                                                  Enzyme                  Compound 6                                            ______________________________________                                        Acetyl CoA Synthetase   0.49                                                  HMG CoA Reductase       1.62                                                  Acyl CoA Cholesterol Acyl Transferase                                                                 1.22                                                  Squalene Cycloase       0.51                                                  Acetyl CoA Carboxylase  1.11                                                  sn-Glycerol-3-Phosphate Acyl Transferase                                                              1.47                                                  Phosphatidylate Phosphohydrolase                                                                      0.91                                                  ______________________________________                                    

Thus, the phosphorus-boron derivatives of the present invention havebeen shown to be potent hypolipidemic agents in rodents, significantlylowering both serum cholesterol and serum triglycerides in mice at a lowdose of 8 mg/kg/day, which is not true for many commercially availablehypolipidemic agents.

These compounds reduced the activities of hepatic de novo enzymesinvolved in the early cytoplasmic synthesis of cholesterol, i.e. acetylCoA synthetase. The rate limiting enzyme for cholesterol synthesis, HMGCoA reductase, was also significantly inhibited. Enzymes involved intriglyceride synthesis, e.g. acetyl CoA carboxylase, regulatory enzymesphosphatidylate phosphohydrolase and sn-glycerol-3-phosphate acyltransferase were also inhibited.

Example 11 Anti-inflammatory Activity of Phosphite-Borane Compounds

CF₁ male mice (≈25g) were administered test drugs at 8 mg/kg in 0.05%Tween® 80-H₂ O intraperitoneally 3 hr. and again 30 min. prior to theinjection of 0.2 ml of 1% carrageenan in 0.9% saline into the plantarsurface of the right hind foot. Saline was injected into the left hindfoot which serves as a base line. After 3 hours, both feet were excisedat the tibiotarsal (ankle)s joint according to the modified method ofWinter (Winter et al, Proc. Soc. Exp. Biol. Med. 1962, 111, 544-547, andHendershot and Forsaith, J. Pharmacol. Exp. Ther. 1970, 175, 435-442).The control mice afforded a 78±3 mg increase in the paw weight. Data arepresented in Table 4 below.

                  TABLE 4                                                         ______________________________________                                        Anti-inflammatory activity of compound 1 & 6 in CF.sub.1 mice at              8 mg/kg.                                                                      Compound  Percent of Control (Induced edema)                                  ______________________________________                                        6         73.9                                                                ______________________________________                                    

While the invention has been described herein with reference toillustrative compounds and specific embodiments of the invention, itwill be appreciated that numerous variations, modifications, and otherembodiments are possible, and accordingly, all such variations,modifications, and embodiments are to be regarded as being within thespirit and scope of the invention.

What is claimed is:
 1. A phosphite-borane compound corresponding to theformula: ##STR16## where: Nucleoside is a natural or syntheticnucleoside connected to the phosphorus via a hydroxyl oxygen;n=1 or 2;each X is independently selected from O and BHR₃ R₄ ; R₁ is selectedfrom H, alkyl, alkylaryl, monovalent metal ions, and ammonium cation; R₂is selected from OR₁ PO₄ ²⁻, OP(O)(OR₅)₂ and N(R₅)₂ and N(R₅)₂, whereinR₅ is independently selected from H, C₁ -C₁₀ linear or branched alkyl,and aryl; R₃ is selected from H, CN, COOH, carboxyl salts, COOR₆ andCONHR₆, wherein R₆ is selected from H, C₁ -C₁₀ alkyl, alkylaryl, andaryl; and R₄ is selected from H and C₁ -C₁₀ alkyl.
 2. A phosphite-boranecompound corresponding to the formula: ##STR17## where: Nucleoside is anatural or synthetic nucleoside connected to the phosphorus via ahydroxyl oxygen;each X is independently selected from O and BHR₃ R₄ ; R₁is selected from H, alkyl, aryl, alkylaryl, monovalent metal ions, andammonium cation; R₂ is selected from OR₁ or N(R₅)₂, wherein R₅ isindependently selected from H, C₁ -C₁₀ linear or branched alkyl, andaryl; R₃ is selected from H and C₁ -C₁₀ alkyl.
 3. A method of making aphosphite-borane compound of the formula: ##STR18## where: Nucleoside isa natural or synthetic nucleoside connected to the phosphorus via ahydroxyl oxygen;n=1 or 2; each X is independently selected from O andBHR₃ R₄ ; R₁ is selected from H, alkyl, alkylaryl, monovalent metalions, and ammonium cation; R₂ is selected from OR₁ PO₄ ²⁻ OP(O)(OR₅)₂and N(R₅)₂, wherein R₅ is independently selected from H, C₁ -C₁₀ linearor branched alkyl, and aryl; R₃ is selected from H, CN, COOH, carboxylsalts, COOR₆ and CONHR₆, wherein R₆ is selected from H, C₁ -C₁₀ alkyl,alkylaryl, and aryl; and R₄ is selected from H and C₁ -C₁₀ alkyl; saidmethod comprising the steps of: phosphitylating a nucleoside moiety toform a phosphoramidite intermediate: transferring boron from a Lewisbase to the phosphoramidite intermediate to form a boronatedphosphoramidite intermediate; de-esterifying the boranatedphosphoramidite intermediate to form a boranophosphoramidate; andreacting the boranophosphoramidate by hydrolysis or by reaction with aphosphate co-reactant selected from the group consisting oforthophosphate and pyrophspate, to yield a correspondingboranophosphate, boranodiphosphate, or α-boranotriphosphate reactionproduct.
 4. A method of making a phosphite-borane compound of theformula: ##STR19## where: Nucleoside is a natural or syntheticnucleoside connected to the phosphorus via a hydroxyl oxygen;n=1 or 2;each X is independently selected from O and BHR₃ R₄ ; R₁ is selectedfrom H, alkyl, alkylaryl, monovalent metal ions, and ammonium cation; R₂is selected from OR₁, PO₄ ²⁻, OP(O)(OR₅)₂ and N(R₅)₂, wherein R₅ isindependently selected from H, C₁ -C₁₀ linear or branched alkyl, andaryl; R₃ is selected from H, CN, COOH, carboxyl salts, COOR₆ and CONHR₆,wherein R₆ is selected from H, C₁ -C₁₀ alkyl, alkylaryl, and aryl; andR₄ is selected from H and C₁ -C₁₀ alkyl; said method comprising thesteps of: transferring borane from a Lewis base-borane adduct to chloro(N,N-dialkyl)alkylphosphoramidite to form a boranophosphorylation agent;boranophosphorylating a nucleoside moiety with the boranophosphorylationagent to form a boranophosphorylated nucleoside; de-esterifying theboranophosphorylated nucleoside to form a boranophosphoramidate; andreacting the boranophosphoramidate by hydrolysis or by reaction with aphosphate co-reactant selected from the group consisting oforthophosphate and pyrophosphate, to yield a correspondingboranophosphate, boranodiphosphate, or α-boranotriphosphate reactionproduct.
 5. A method for the preparation of a phosphite-borane compoundof the formula: ##STR20## where: Nucleoside is a natural or syntheticnucleoside connected to the phosphorus via a hydroxyl oxygen;n=1 or 2;each X independently selected from O and BHR₃ R₄ ; R₁ is selected fromH, alkyl, alkylaryl, monovalent metal ions and ammonium cation; R₂ isselected from OR₁, PO₄ ²⁻, OP(O)(OR₅)₂ and N(R₅)₂, wherein R₅ isindependently selected from H, C₁ -C₁₀ linear or branched alkyl, andaryl; R₃ is selected from H, CN, COOH, carboxyl salts, COOR₆ and CONHR₆,wherein R₆ is selected from H, C₁ -C₁₀ alkyl, alkylaryl, and aryl; andR₄ is selected from H and C₁ -C₁₀ alkyl, said method comprising thesteps of:providing a boranophosphoramidite of the formula; ##STR21##de-esterifying the boranophosphoramidite to form aboranophosphoramidate; and reacting the boranophosphoramidate byreaction with a phosphate co-reactant selected from the group consistingof orthophosphate and pyrophosphate, to yield a correspondingboranophosphate, borandiphosphate, or α-boranotriphosphate reactionproduct.
 6. A method of preparing a phosphite-borane compound of theformula: ##STR22## where: Nucleoside_(a) is a natural or syntheticnucleoside connected to the phosphorus via a hydroxyl oxygen;n=1 or 2;each X is independently selected from O and BHR₃ R₄ ; R₁ is selectedfrom H, alkyl, alkylaryl, monovalent metal ions and ammonium cation; R₂is selected from OR₁, PO₄ ²⁻, OP(O)(OR₅)₂ and N(R₅)₂, wherein R₅ isindependently selected from H, C₁ -C₁₀ linear or branched alkyl, andaryl; R₃ is selected from H, CN, COOH, carboxyl salts, COOR₆ and CONHR₆,wherein R₆ is selected from H, C₁ -C₁₀ alkyl, alkylaryl, and aryl; andR₄ is selected from H and C₁ -C₁₀ alkyl, said method comprising:reacting a compound of the formula ##STR23## in the presence of acondensing agent to yield the phosphite-borane compound; ##STR24##wherein Nucleoside is a phosphorus-containing nucleoside moietyincluding Nucleoside_(a).
 7. A method according to claim 3, wherein thenucleoside moiety is selected from the group consisting ofnucleo-side-phosphate and nucleoside diphosphate.